Alzheimer's disease (“AD”) is a devastating neurodegenerative disease that affects millions of elderly patients worldwide. AD is characterized clinically by progressive loss of memory, orientation, cognitive function, judgement and emotional stability. With increasing age, the risk of developing AD increases exponentially, so that by age 85 some 20-40% of the population is affected. Memory and cognitive function deteriorate rapidly within the first 5 years after diagnosis of mild to moderate impairment, and death due to disease complications is an inevitable outcome. AD is the most common cause of nursing home admittance in the United States; hence, in addition to the morbidity and mortality experienced by the patient, there are considerable economic and emotional burdens placed on the family, caregivers and society at large. The only recognized treatment currently available for AD is acetylcholinesterase inhibitors, which merely treat the symptoms of cognitive impairment. No method for prevention or treatment of the pathophysiology of AD is currently available.
Diagnosis of AD is based mainly on subjective assessments of memory and cognitive function. Definitive diagnosis can only be made post-mortem, based on histopathological examination of brain tissue from the patient. Two histological hallmarks of AD are the occurrence of neurofibrillar tangles of hyperphosphorylated tau protein and of proteinaceous amyloid plaques, both within the cerebral cortex of AD patients. The amyloid plaques are composed mainly of a peptide of 39 to 42 amino acids designated beta-amyloid, also referred to as β-amyloid, amyloid beta, Aβ, βAP, β/A4; and referred to herein as beta-amyloid and Aβ. It is now clear that the Aβ peptide is derived from a type 1 integral membrane protein, termed beta amyloid precursor protein (also referred to as “β-APP” and “APP”) through two sequential proteolytic events. First, the APP is hydrolyzed at a site N-terminal of the transmembrane alpha helix by a specific proteolytic enzyme referred to as β-secretase. The soluble N-terminal product of this cleavage event diffuses away from the membrane, leaving behind the membrane-associate C-terminal cleavage product, referred to as C99. The protein C99 is then further hydrolyzed within the transmembrane alpha helix by a specific proteolytic enzyme referred to as γ-secretase. This second cleavage event liberates the Aβ peptide and leaves a membrane-associated “stub”. The Aβ peptide thus generated is secreted from the cell into the extracellular matrix where it eventually forms the amyloid plaques associated with AD.
Several lines of evidence suggest that abnormal accumulation of Aβ plays a key role in the pathogenesis of AD. First, Aβ is the major protein component of amyloid plaques. Second, Aβ is neurotoxic and may be causally linked to the neuronal death associated with AD. Third, missense DNA mutations at several positions within the APP protein can be found in affected members but not unaffected members of several families with a genetically determined (familial) form of AD. For example, one familial form of AD is linked to a pair of mutations, referred to as the “Swedish mutations”, that are immediately proximal to the site of β-secretase-mediated hydrolysis of APP (Mullan et al., (1992) Nature Genet. 1:345-347). Patients bearing the Swedish mutant form of APP develop AD at a much earlier age (typically within the fourth decade of life) and likewise progress to severe dementia at a much earlier age. Histopathological examination of the brains of patients suffering from the “Swedish mutant” form of familial AD is identical to that of brains from patients suffering from non-familial, sporadic forms of the disease. It is therefore hypothesized that halting the production of Aβ will prevent and/or reduce the neurodegeneration and other pathologies of AD. One method of halting Aβ production would be to administer specific inhibitors of one or both of the proteolytic enzymes involved in APP processing, namely, β-secretase and γ-secretase. The molecular identity of the protein responsible for γ-secretase activity has not yet been determined, although there is a preponderance of data suggesting a role for the proteins presenilin-1 and presenilin-2 in this enzymatic action. Nevertheless, compounds that inhibit the action of γ-secretase, and thus inhibit Aβ production in cell culture have been identified by several groups.
Recently the molecular identity of the protein responsible for β-secretase activity has been determined and this protein is commonly referred to as BACE (for Beta-site APP Cleaving Enzyme). This enzyme is a type 1 membrane protein that folds into an extra-membranous globular catalytic domain that is tethered to the membrane by a single alpha helix. The catalytic domain of BACE contains the canonical signature motifs for an aspartyl protease, and the enzymatic activity of recombinant versions of the catalytic domain of human BACE is consistent with this designation. It is well known that aspartyl proteases can be effectively inhibited by small molecules and peptides that bind to, and hence block, the site on the enzyme molecule at which the chemical transformations of the substrate molecule takes place. This site of chemical reactivity is commonly referred to as the enzyme active site. For aspartyl proteases this site contains the two chemically reactive aspartic acid residues from which this class of enzymes derive its name. During the course of enzymatic action on the substrate molecule, the enzyme goes through an intermediate state in which the carbonyl carbon of the hydrolyzable amide bond of the substrate forms four coordinate bonds, engaging the active site aspartic acid residues of the enzyme.
A common strategy for inhibiting aspartyl proteases is to prepare a small peptide of amino acid composition similar to the substrate molecule but replacing the hydrolyzable amide bond with a chemical group that mimics the four coordinate carbon intermediate species just described. It is well known that chemical groups such as statines, hydroxyethylenes, hydroxyethylamines and similar structures are very effective for this purpose. Indeed, peptidic inhibitors of BACE, incorporating statine and hydroxyethylene structures have been reported. Recently the 3-dimensional structure of the catalytic domain of human BACE in complex with a hydroxyethylene-based peptidic inhibitor referred to as OM99-2 has been solved by the methods of x-ray crystallography. The resulting structure confirmed that the inhibitor binds within the enzyme active site, engaging the active site aspartic acid residues as expected. Hence, active site-directed inhibitors of BACE can be designed and may prove useful as pharmacological agents for the treatment of AD. Historically, however, it has proved difficult to develop molecules of pharmacological utility based on active site-directed inhibitors of aspartyl proteases. While very potent inhibitors have been identified in vitro, active site-directed inhibitors of aspartyl proteases may present in vivo issues of oral bioavailability and pharmacokinetic half-life.
In addition to the active site, some proteolytic enzymes contain additional binding pockets that engage the substrate protein at locations distal to the site of chemical transformation. These binding pockets are referred to as exosites and can contribute significantly to the stabilization of the enzyme-substrate binary complex by providing important structural determinants of interaction. Additionally, exosites on some proteolytic enzymes can act as allosteric regulators of enzyme activity, so that binding interactions at the exosite are transmitted through conformational changes of the enzyme to the active site, where structural changes can augment or diminish the chemical reactivity of the active site. In some cases molecules have been identified that bind to an exosite, rather than the active site, of proteolytic enzymes and these have proved to be effective inhibitors of enzymatic action. Hence, exosites represent an alternative target for inhibitory ligand binding to proteolytic enzymes. Because the exosites are distinct from the active sites of these enzymes, the nature of the molecules that bind to the exosites can be very different from active site-directed inhibitors. In favorable cases, the nature of the molecules binding to the exosites are more pharmacologically tractable relative to the active site-directed inhibitors of the same enzyme.